Davin-Regli, R. Chollet, J. Bredin, J. Chevalier, F.
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Davin-Regli, R. Chollet, J. Bredin, J. Chevalier, F. Lepine, J. Objectives : In order to characterize the mechanism involved in parabens resistance, we studied 13 Enterobacter gergoviae collected from diverse cosmetic formulations containing parabens as preservatives and 10 isolates from clinical or industrial sources. To study antibiotic and paraben susceptibility, the standard disc diffusion method and the 2-fold dilution method in Luria—Bertani medium were used.
Characterization of porins was performed using immunodetection with polyclonal antibodies. Results : Epidemiological typing confirmed that most of the cosmetic formulations were contaminated by unrelated strains. All of the E. Conclusions : This is the first report showing the role of active efflux in the parabens resistance in E. Paraben efflux seems to be regulated by a mar -independent process in E.
Parabens are used as antimicrobial preservatives, particularly in cosmetic products; furthermore, they are likely to have multiple targets and biological effects. An esterase named PrbA, which hydrolyses high concentrations of parabens into 4-hydroxybenzoic acid, has recently been identified. Enterobacter gergoviae is rarely isolated in clinical laboratories and it is generally susceptible to antibiotics however, some cosmetic laboratories were concerned about the contamination of their different cosmetic formulations, containing a combination of parabens as preservatives, by E.
From this study it may be concluded that most of the cosmetics were contaminated by unrelated strains showing a natural paraben efflux mechanism that does not affect antibiotics.
The marRAB operon of E. Considering that E. Over the period —, 13 E. One or more parabens in combination were present in concentrations between 0. We selected 10 other E. Isolates were confirmed to be E.
Ea 27 strain is a multidrug-resistant MDR clinical Enterobacter aerogenes , used as a control for its efflux resistance mechanism, that overexpresses the AcrAB-TolC efflux system.
The MICs of methylparaben were determined on a methylparaben gradient: 6 each strain was spread onto successive gradient LB agar plates, containing concentrations ranging from 0. MICs of methylparaben were confirmed by a standard 2-fold dilution method in LB medium. Three successive subcultures preceded antibiotic susceptibility evaluation by the disc diffusion method. Each result was an average of two experiments.
Exponential bacterial cells grown in LB broth were collected. The nitrocellulose membranes were then incubated in the same buffer supplemented with 0. Polyclonal antibodies directed against the E. It has been shown that propylparaben is able to open bacterial mechanosensitive channels, allowing the leakage of cytoplasmic content. The potassium efflux measurements were carried out after addition of propylparaben 0.
Preparation of DNA was performed using the hexadecyltrimethylammonium bromide method. The identical strains within each pair were isolated from the same laboratory, but the year of isolation was different in two of the three cases. Polyclonal antibodies allowed us to investigate the presence of porins in the various E. Antigenic related porins were observed in the 23 strains, irrespective of the level of paraben resistance data not shown. The 23 strains were susceptible to the antibiotics tested, except the clinical strains E.
The effect on antibiotic resistance of subculturing in the presence of propylparaben or salicylate, a well-known mar inducer, was investigated. After subculturing in the presence of salicylate, which is an o- hydroxybenzoic acid structurally analogous to parabens, all strains except EG2 presented a diminution of drug susceptibilities Table 1. Conversely, propylparaben, which is structurally analogous to salicylate, is not able to activate the MDR cascade mediated by marA , as demonstrated by the absence, or very low modifications, of antibiotic susceptibility.
These results suggest little effect of parabens on the induction of MDR. Comparison of the inhibition zone sizes in the six E.
Paper discs were used 6 mm diameter. Transformation by the plasmid p9 induced a pleotropic diminution of antibiotic susceptibility, confirming the existence of a MarA-mediated response in E.
However, little effect was observed on methylparaben susceptibility Table 2. MICs of methylparaben for Ea 27, E. The six E. Influence of MarA- Eco on methylparaben susceptibility was investigated in strains transformed by p9 expressing MarA- Eco. Esterase activity: expressed as the percentage of ppm of methylparaben 5. Among the 23 E. These results suggest the existence of an additional resistance mechanism. Pairs of primers gave specific amplification products that allowed us to obtain sequences of bp exhibiting a putative COOH-terminal part of marC , marO , marR , marA and marB genes.
For the two strains studied, an identical sequence was found. The amino acid sequences varied in length, with a protein of residues in E. Divergence of amino acid sequences between the different species was higher than that deduced for MarA.
EG7 was 3 times more resistant than EG4 to methylparaben: this could indicate that esterase expression in EG7 has a protective action towards methylparaben activity. We have observed that addition of propylparaben to E.
Here we observed that E. These results indicated that resistance mechanisms detected in EG7, such as efflux pump and esterase expression present in the periplasm, efficiently protected EG7 membrane from propylparaben activity.
Valkova et al. Thus, esterase alone did not confer a sufficiently rapid and protective action against the toxic effect of methylparaben on membranes. In bacteria, additional efflux of propylparaben generates a synergic effect on resistance, probably by decreasing the periplasmic paraben concentration.
No direct relationship was observed between paraben and antibiotic resistance. This study confirmed that E. It is independent of antibiotic efflux, as seen in Ea 27, which preserves methylparaben susceptibility, despite overexpression of the AcrAB-TolC efflux pump.
Considering that i efflux pumps with narrow substrate specificity exist even for antibiotics, as demonstrated for telithromycin in E. Although the origin of E. This work is dedicated to Claude Bollet, who initiated the study but passed away in August We thank M. Mounier and F. Safety assessment of propylparaben: a review of the published literature. Food Chem Toxicol ; 39 : — J Bacteriol ; : —7. Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes.
Microbiology ; : —9. Antimicrob Agents Chemother ; 46 : —7. Molecular epidemiology of Enterobacter aerogenes acquisition: one-year prospective study in two intensive care units. J Clin Microbiol ; 34 : — Szybalski W. Microbial selection. Gradient plate technique for study of bacterial resistance. Science ; : 46 —8. Current Protocols in Molecular Biology. The effects of parabens on the mechanosensitive channels of E. Eur Biophys J ; 34 : — Propyl paraben induces potassium efflux in Escherichia coli.
J Antimicrob Chemother ; 55 : —5. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother ; 41 : — The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli. Antimicrob Agents Chemother ; 48 : —4. Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve? J Bacteriol ; : — Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA.
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Enterobacter Gergoviae Adaptation to Preservatives Commonly Used in Cosmetic Industry
Objective: To investigate the natural susceptibility to 69 antimicrobial agents of Enterobacter strains comprising E. Methods: Minimal inhibitory concentrations MICs were determined with a microdilution procedure in Isosensitest broth and cation-adjusted Mueller-Hinton broth. Natural resistance was found to penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-related differences in natural susceptibility were found to some beta-lactams, azithromycin and fosfomycin. Whereas E.
Laurie Kundrat April 27, 3 Comments. Pluralibacter gergoviae can cause big headaches in cosmetic industry laboratories. It is an opportunistic pathogen that has repeatedly been isolated from personal care products. Recently, 15, tubes of a popular skin cream product were recalled due to contamination with the… Read more.
Objective: The aim of this study was to obtain a better understanding regarding the origin of recurrent contamination by Enterobacter gergoviae in diverse cosmetic formula. We studied 65 isolates collected from various sources clinical, food, cosmetics. Evaluation of susceptibility to preservatives currently used in cosmetics for a representative panel of collection strains was measured. Preservative efficacy was evaluated by minimum inhibitory concentrations and minimum bactericidal concentrations MBCs. Results: Eighty per cent of isolates was unrelated.
Enterobacter is a genus of common Gram-negative , facultatively anaerobic , rod-shaped , non-spore-forming bacteria of the family Enterobacteriaceae. It is the type genus of the order Enterobacterales. The urinary and respiratory tracts are the most common sites of infection. The genus Enterobacter is a member of the coliform group of bacteria. It does not belong to the fecal coliforms or thermotolerant coliforms group of bacteria, unlike Escherichia coli , because it is incapable of growth at Some of them showed quorum sensing properties as reported before.